From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Abstract

Voltage-gated sodium channels are responsible for the rising phase of the action potential in the membranes of neurons and most electrically excitable cells. Exploration of the molecular properties of voltage-gated ion channels began twenty years ago this past January with report of the discovery of the sodium channel protein by neurotoxin labeling methods (1Agnew W.S Moore A.C Levinson S.R Raftery M.A Identification of a large molecular weight peptide associated with a tetrodotoxin binding proteins from the electroplax of Electrophorus electricus.Biochem. Biophys. Res. Commun. 1980; 92: 860-866Crossref PubMed Scopus (61) Google Scholar, 6Beneski D.A Catterall W.A Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin.Proc. Natl. Acad. Sci. USA. 1980; 77: 639-643Crossref PubMed Scopus (119) Google Scholar). This review briefly recounts the early biochemical studies of sodium channels, focuses primarily on the emerging view of the molecular mechanisms of sodium channel function and regulation, and gives a perspective for future research on the expanding family of sodium channel proteins. Discoveries of sodium channel diseases, new sodium channel genes, new modes of modulation of sodium channel function, and new forms of sodium channel protein–protein interactions greatly expand the possible roles for sodium channels in neuronal function and regulation. Sodium currents were first recorded by Hodgkin and Huxley, who used voltage clamp techniques to demonstrate the three key features that have come to characterize the sodium channel: (1) voltage-dependent activation, (2) rapid inactivation, and (3) selective ion conductance (44Hodgkin A.L Huxley A.F A quantitative description of membrane current and its application to conduction and excitation in nerve.J. Physiol. 1952; 117: 500-544Crossref PubMed Scopus (12310) Google Scholar). Detailed analysis of sodium channel function during the 1960s and 1970s using the voltage clamp method applied to invertebrate giant axons and vertebrate myelinated nerve fibers yielded mechanistic models for sodium channel function (2Armstrong C.M Sodium channels and gating currents.Physiol. Rev. 1981; 61: 644-682Crossref PubMed Scopus (343) Google Scholar, 42Hille B Ionic Channels of Excitable Membranes. First Edition. Sinauer Associates, Inc, Sunderland, MA1984Google Scholar). These functional studies predicted that sodium channels would be rare membrane proteins, difficult to identify and to isolate from the many other proteins of excitable membranes. During the 1970s, a new line of research emerged, focussing on development of methods for molecular analysis of sodium channels. Biochemical methods for measurement of ion flux through sodium channels, high affinity binding of neurotoxins to sodium channels, and detergent solubilization and purification of sodium channel proteins labeled by neurotoxins were progressively developed (reviewed in 79Ritchie J.M Rogart R.B The binding of saxitoxin and tetrodotoxin to excitable tissue.Rev. Physiol. Biochem. Pharmacol. 1977; 79: 1-49Crossref PubMed Google Scholar, 12Catterall W.A Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes.Annu. Rev. Pharmacol. Toxicol. 1980; 20: 15-43Crossref PubMed Scopus (981) Google Scholar). These biochemical approaches led to discovery of the sodium channel protein in 1980. Photoaffinity labeling with a photoreactive derivative of an α-scorpion toxin identified the principal α subunit (260 kDa) and the auxiliary β1 subunit (36 kDa) of brain sodium channels (6Beneski D.A Catterall W.A Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin.Proc. Natl. Acad. Sci. USA. 1980; 77: 639-643Crossref PubMed Scopus (119) Google Scholar). Immediately thereafter, partial purification of tetrodotoxin binding proteins from electric eel electroplax revealed a correlation between tetrodotoxin binding activity and a protein of ∼270 kDa (1Agnew W.S Moore A.C Levinson S.R Raftery M.A Identification of a large molecular weight peptide associated with a tetrodotoxin binding proteins from the electroplax of Electrophorus electricus.Biochem. Biophys. Res. Commun. 1980; 92: 860-866Crossref PubMed Scopus (61) Google Scholar). Subsequent purification studies showed that the sodium channel from mammalian brain is a complex of α (260 kDa), β1 (36 kDa), and β2 (33 kDa) subunits (36Hartshorne R.P Catterall W.A Purification of the saxitoxin receptor of the sodium channel from rat brain.Proc. Natl. Acad. Sci. USA. 1981; 78: 4620-4624Crossref PubMed Scopus (132) Google Scholar, 37Hartshorne R.P Messner D.J Coppersmith J.C Catterall W.A The saxitoxin receptor of the sodium channel from rat brain evidence for two nonidentical beta subunits.J. Biol. Chem. 1982; 257: 13888-13891PubMed Google Scholar), the tetrodotoxin-binding component of the sodium channel purified from eel electroplax is a single protein of 270 kDa (65Miller J.A Agnew W.S Levinson S.R Principal glycopeptide of the tetrodotoxin/saxitoxin binding protein from Electrophorous electricus isolation and partial chemical and physical characterization.Biochemistry. 1983; 22: 462-470Crossref PubMed Scopus (155) Google Scholar), and the sodium channel from skeletal muscle is a complex of α and β1 subunits (4Barchi R.L Protein components of the purified sodium channel from rat skeletal sarcolemma.J. Neurochem. 1983; 36: 1377-1385Crossref Scopus (80) Google Scholar). Reconstitution of neurotoxin-activated ion flux through purified sodium channels of known subunit composition from brain and skeletal muscle revealed that they contain a functional pore (100Talvenheimo J.A Tamkun M.M Catterall W.A Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain.J. Biol. Chem. 1982; 257: 11868-11871Abstract Full Text PDF PubMed Google Scholar, 52Kraner S.D Tanaka J.C Barchi R.L Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes.J. Biol. Chem. 1985; 260: 6341-6347PubMed Google Scholar), and fusion into planar bilayer membranes demonstrated voltage-dependent activation (38Hartshorne R.P Keller B.U Talvenheimo J.A Catterall W.A Montal M Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.Proc. Natl. Acad. Sci. USA. 1985; 82: 240-244Crossref PubMed Scopus (100) Google Scholar, 31Furman R.E Tanaka J.C Mueller P Barchi R.L Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.Proc. Natl. Acad. Sci. USA. 1986; 83: 488-492Crossref PubMed Scopus (21) Google Scholar). Together, these studies showed that the purified sodium channel protein contained the essential elements for ion conduction and voltage-dependent gating and opened the way for more detailed molecular analysis of the channel protein. Using both oligonucleotides encoding short segments of the electric eel electroplax sodium channel as well as antibodies directed against the channel protein, 67Noda M Shimizu S Tanabe T Takai T Kayano T Ikeda T Takahashi H Nakayama H Kanaoka Y Minamino N et al.Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence.Nature. 1984; 312: 121-127Crossref PubMed Scopus (930) Google Scholar isolated cDNAs encoding the entire polypeptide from expression libraries of electroplax mRNA. Their work gave the initial insight into the primary structure of a voltage-gated ion channel. The deduced amino acid sequence revealed a large protein with four internally homologous domains, each containing multiple potential α-helical transmembrane segments (Figure 1B). The structural motif of the sodium channel homologous domains is the building block of the voltage-gated calcium and potassium channels and a large family of related ion channels, including the cyclic nucleotide-gated channels, calcium-activated potassium channels, and trp-like calcium channels. The wealth of information contained in this deduced primary structure revolutionized research on the voltage-gated ion channels. Although the primary structure of the eel electroplax sodium channel provided the template for molecular analysis, successful functional expression of the sodium channel required cloning cDNAs from rat brain. RNA encoding the α subunit proved sufficient for functional expression of sodium currents in Xenopus oocytes (34Goldin A.L Snutch T Lubbert H Dowsett A Marshall J Auld V Downey W Fritz L.C Lester H.A Dunn R Catterall W.A Davidson N Messenger RNA coding for only the α subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.Proc. Natl. Acad. Sci. 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The β1 and β2 subunits of sodium channels have similar overall structures but are not closely related in amino acid sequence. Both β subunits have a large, glycosylated extracellular domain, a single transmembrane segment, and a small intracellular domain (Figure 1; 45Isom L.L De Jongh K.S Patton D.E Reber B.F.X Offord J Charbonneau H Walsh K Goldin A.L Catterall W.A Primary structure and functional expression of the β1 subunit of the rat brain sodium channel.Science. 1992; 256: 839-842Crossref PubMed Scopus (571) Google Scholar, 46Isom L.L Ragsdale D.S De Jongh K.S Westenbroek R.E Reber B.F.X Scheuer T Catterall W.A Structure and function of the β2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM-motif.Cell. 1995; 83: 433-442Abstract Full Text PDF PubMed Scopus (381) Google Scholar). Application of molecular modeling to the sodium channel led to a remarkably accurate prediction of the two-dimensional folding pattern of the α subunit, even before any experimental analysis of structure and function was available (35Guy H.R Seetharamulu P Molecular model of the action potential sodium channel.Proc. Natl. Acad. Sci. USA. 1986; 508: 508-512Crossref Scopus (403) Google Scholar). This analysis predicted six α-helical transmembrane segments (S1–S6) in each of the four homologous domains (I–IV) and a reentrant loop that dipped into the transmembrane region of the protein between transmembrane segments S5 and S6 and formed the outer pore (Figure 1B). Relatively large extracellular loops were predicted in each homologous domain, connecting either the S5 or S6 transmembrane segments to the membrane-reentrant loop. Even larger intracellular loops were predicted to connect the four homologous domains, and large N-terminal and C-terminal domains were also predicted to be intracellular. Subsequent work on sodium, calcium, and potassium channels is consistent with the general features of this early model. Comparison of the primary structures of the auxiliary β1 and β2 subunits to those of other proteins revealed a probable structural relationship to the family of proteins that contain immunoglobulin-like folds (46Isom L.L Ragsdale D.S De Jongh K.S Westenbroek R.E Reber B.F.X Scheuer T Catterall W.A Structure and function of the β2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM-motif.Cell. 1995; 83: 433-442Abstract Full Text PDF PubMed Scopus (381) Google Scholar). The extracellular domains of the β1 and β2 subunits are predicted to fold in a similar manner as myelin protein p0, whose structure is known (Figure 1B; 87Shapiro L Doyle J.P Hensley P Colman D.R Hendrickson W.A Crystal structure of the extracellular domain from Po, the major structural protein of peripheral nerve myelin.Neuron. 1996; 17: 435-449Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The immunoglobulin-like fold is a sandwich of two β sheets held together by hydrophobic interactions. To date, the β1 and β2 subunits are unique among ion channel subunits in containing immunoglobulin-like folds. Just as the high affinity and specificity of neurotoxins led to discovery of the sodium channel protein, the pore blockers tetrodotoxin and saxitoxin also led directly to identification of the outer pore and selectivity filter. Voltage clamp studies provided a model of tetrodotoxin and saxitoxin as plugs of the selectivity filter in the outer pore of sodium channels (42Hille B Ionic Channels of Excitable Membranes. First Edition. Sinauer Associates, Inc, Sunderland, MA1984Google Scholar). Mutational analysis identified glutamate 387 in the membrane-reentrant loop in domain I as a crucial residue for tetrodotoxin and saxitoxin binding (70Noda M Suzuki H Numa S Stühmer W A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II.FEBS. Lett. 1989; 259: 213-216Abstract Full Text PDF PubMed Scopus (258) Google Scholar), and subsequent studies revealed a pair of important amino acid residues, mostly negatively charged, in analogous positions in all four domains (Figure 1B, small white circles; 102Terlau H Heinemann S.H Stühmer W Pusch M Conti F Imoto K Numa S Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II.FEBS Lett. 1991; 293: 93-96Abstract Full Text PDF PubMed Scopus (347) Google Scholar). These amino acid residues were postulated to form outer and inner rings that serve as the receptor site for tetrodotoxin and saxitoxin and as the selectivity filter in the outer pore of sodium channels. This view derives strong support from the finding that sodium channels can be made calcium selective by exchanging the amino acid residues in the inner ring from DEKA in domains I–IV to their counterparts EEEE in calcium channels (40Heinemann S.H Terlau H Stühmer W Imoto K Numa S Calcium channel characteristics conferred on the sodium channel by single mutations.Nature. 1992; 356 (b): 441-443Crossref PubMed Scopus (602) Google Scholar). Mutations in this postulated ring of four amino acid residues have strong effects on selectivity for organic and inorganic monovalent cations, in agreement with the idea that they form the selectivity filter (e.g., 85Schlief T Schönherr R Imoto K Heinemann S.H Pore properties of rat brain II sodium channels mutated in the selectivity filter domain.Eur. Biophys. J. 1996; 25: 75-91Crossref PubMed Scopus (92) Google Scholar, 98Sun Y.M Favre I Schild L Moczydlowski E On the structural basis for size-selective permeation of organic cations through the voltage-gated sodium channel—effect of alanine mutations at the DEKA locus on selectivity, inhibition by Ca2+ and H+, and molecular sieving.J. Gen. Physiol. 1997; 110: 693-715Crossref PubMed Scopus (114) Google Scholar). These conclusions about the structure of the outer pore of sodium channels also derive strong support from work on potassium channels. A corresponding pore loop was identified in potassium channels and shown to control ion conductance and selectivity (64Miller C 1990 annus mirabilis for potassium channels.Science. 1991; 252: 1092-1096Crossref PubMed Scopus (189) Google Scholar), and the three-dimensional structure of a bacterial potassium channel reveals a narrow ion selectivity filter formed by the pore loop from its four identical subunits (28Doyle D.A Cabral J.M Pfuetzner R.A Kuo A.L Gulbis J.M Cohen S.L Chait B.T MacKinnon R The structure of the potassium channel molecular basis of K+ conduction and selectivity.Science. 1998; 280: 69-77Crossref PubMed Scopus (5299) Google Scholar). The two rings of amino acid residues that form the sodium channel selectivity filter are illustrated superimposed on the potassium channel structure in Figure 2 (red). However, in the potassium channel, the backbone carbonyl groups of the peptide bonds of three consecutive amino acid residues form the narrow part of the selectivity filter, in contrast to the suggestion from mutagenesis studies of sodium channels that the carboxyl side chains of glutamate and aspartate residues interact with permeant ions and determine ion selectivity. It will be of great interest to learn how the structure of the outer pore of potassium channels is modified to yield the different ion selectivity of sodium and calcium channels. All sodium channels studied to date have similar permeation properties and are therefore expected to have similar selectivity filters. However, cardiac sodium channels bind tetrodotoxin with 200-fold lower affinity because of a change of a tyrosine or phenylalanine, which is located two positions preceding the first pore glutamate in domain I in the brain and skeletal muscle channels, to cysteine in the cardiac sodium channel (3Backx P.H Yue D.T Lawrence J.H Marban E Tomaselli G.F Molecular localization of an ion-binding site within the pore of mammalian sodium channels.Science. 1992; 257: 248-251Crossref PubMed Scopus (236) Google Scholar, 39Heinemann S.H Terlau H Imoto K Molecular basis for pharmacological differences between brain and cardiac sodium channels.Pflugers Arch. 1992; 422 (a): 90-92Crossref PubMed Scopus (93) Google Scholar, 84Satin J Kyle J.W Chen M Bell P Cribbs L.L Fozzard H.A Rogart R.B A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties.Science. 1992; 256: 1202-1205Crossref PubMed Scopus (323) Google Scholar). Serine in this position in some peripheral nervous system sodium channels causes even larger decreases in tetrodotoxin binding affinity (90Sivilotti L Okuse K Akopian A.N Moss S Wood J.N A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS.FEBS Lett. 1997; 409: 49-52Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, a single amino acid residue in the selectivity filter in domain I determines the affinity of different sodium channel types for the pore blocker tetrodotoxin. Cadmium is a high-affinity blocker of cardiac sodium channels but not of brain or skeletal muscle sodium channels because of its interaction with this cysteine residue (3Backx P.H Yue D.T Lawrence J.H Marban E Tomaselli G.F Molecular localization of an ion-binding site within the pore of mammalian sodium channels.Science. 1992; 257: 248-251Crossref PubMed Scopus (236) Google Scholar, 84Satin J Kyle J.W Chen M Bell P Cribbs L.L Fozzard H.A Rogart R.B A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties.Science. 1992; 256: 1202-1205Crossref PubMed Scopus (323) Google Scholar). Analysis of the voltage dependence of cadmium block suggests that this ion passes 20% of the way through the membrane electrical field in reaching its binding site formed by this cysteine residue (3Backx P.H Yue D.T Lawrence J.H Marban E Tomaselli G.F Molecular localization of an ion-binding site within the pore of mammalian sodium channels.Science. 1992; 257: 248-251Crossref PubMed Scopus (236) Google Scholar). Thus, this residue may be ∼20% of the way through the electrical field within the selectivity filter in the pore of the sodium channel. Although the pore structure of ion channels is most often considered to be static, substituted cysteine mutagenesis and cross-linking experiments suggest that the pore loops of sodium channels are asymetrically organized and dynamic on the millisecond time scale of sodium channel gating (7Benitah J.P Chen Z.H Balser J.R Tomaselli G.F Marban E Molecular dynamics of the sodium channel pore vary with gating interactions between P-segment motions and inactivation.J. Neurosci. 1999; 19: 1577-1585PubMed Google Scholar). Cysteine residues substituted in analogous pore positions in domains I–IV are at different positions in the electric field. The domain II pore loop is most superficial, domains I and III intermediate, and domain IV most internal, as judged by the voltage dependence of cadmium block of the mutant channels (24Chiamvimonvat N Pérez-García M.T Ranjan R Marban E Tomaselli G.F Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis.Neuron. 1996; 16: 1037-1047Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Cross-linking of pairs of substituted cysteine residues reveals unexpected flexibility of the pore regions, allowing formation of disulfide bonds between both distant and closely apposed amino acid residues (73Pérez-García M.T Chiamvimonvat N Marban E Tomaselli G.F Structure of the sodium channel pore revealed by serial cysteine mutagenesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 300-304Crossref PubMed Scopus (107) Google Scholar). These cross-linking reactions are enhanced by activation of sodium channels with repetitive pulses, suggesting that increased motion during gating brings potentially reactive cysteine residues close together to allow disulfide bond formation (7Benitah J.P Chen Z.H Balser J.R Tomaselli G.F Marban E Molecular dynamics of the sodium channel pore vary with gating interactions between P-segment motions and inactivation.J. Neurosci. 1999; 19: 1577-1585PubMed Google Scholar). Movement of the selectivity filter region on the millisecond time scale of channel gating is therefore likely. An additional important question is whether these regions can also move on the microsecond time scale of ion conductance. Voltage clamp studies led to the conclusion that local anesthetics enter from the intracellular side and bind in the inner pore of sodium channels (42Hille B Ionic Channels of Excitable Membranes. First Edition. Sinauer Associates, Inc, Sunderland, MA1984Google Scholar), and similar work revealed analogous intracellular block of calcium and potassium channels. As for the outer pore, the first indication that the S6 segments form the inner pore of the voltage-gated ion channels came from locating a pore-blocker receptor site—in this case the phenylalkylamine receptor site of L-type calcium channels (96Striessnig J Glossmann H Catterall W.A Identification of a phenylalkylamine binding region within the α1 subunit of skeletal muscle Ca2+ channels.Proc. Natl. Acad. Sci. USA. 1990; 87: 9108-9112Crossref PubMed Scopus (136) Google Scholar). Photoaffinity labeling with these high-affinity, intracellular pore blockers showed that only the IVS6 segment of the calcium channel α1 subunit was labeled. Subsequently, mutagenesis studies of sodium channels revealed the local anesthetic receptor site in an analogous position in the sodium channel (77Ragsdale D.S McPhee J.C Scheuer T Catterall W.A Molecular determinants of state-dependent block of Na+ channels by local anesthetics.Science. 1994; 265: 1724-1728Crossref PubMed Scopus (690) Google Scholar). High-affinity binding of local anesthetics to the inactivated state of sodium channels requires two critical amino acid residues, Phe-1764 and Tyr-1771 in brain type IIA channels, which are located on the same side of the IVS6 transmembrane segment two α-helical turns apart (Figure 2, Phe-1764 and Tyr-1771 in blue, etidocaine in purple). It is likely that the tertiary amino group of local anesthetics interacts with Phe-1764, which is located more deeply in the pore, and that the aromatic moiety of the local anesthetics interacts with Tyr-1771, which is located nearer to the intracellular end of the pore (Figure 2, blue residues). Subsequent work in several laboratories has shown that sodium channel blocking drugs of diverse structure that are used as antiarrhythmic drugs and as anticonvulsants also interact with the same site as local anesthetics but make additional interactions with other nearby amino acid residues as well. Substituted tetraethylammonium derivatives, which are intracellular blockers of certain potassium channels, interact with similar amino acid residues in the S6 transmembrane segment (25Choi K.L Mossman C Aubé J Yellen G The internal quaternary ammonium receptor site of Shaker potassium channels.Neuron. 1993; 10: 533-541Abstract Full Text PDF PubMed Scopus (218) Google Scholar). These amino acid residues are located in an aqueous cavity within the pore of the Kcsa bacterial potassium channel (28Doyle D.A Cabral J.M Pfuetzner R.A Kuo A.L Gulbis J.M Cohen S.L Chait B.T MacKinnon R The structure of the potassium channel molecular basis of K+ conduction and selectivity.Science. 1998; 280: 69-77Crossref PubMed Scopus (5299) Google Scholar), providing additional support for this location of the local anesthetic receptor site in sodium channels. The voltage dependence of activation of the sodium channel and other voltage-gated ion channels derives from the outward movement of gating charges in response to changes in the membrane electric field (44Hodgkin A.L Huxley A.F A quantitative description of membrane current and its application to conduction and excitation in nerve.J. Physiol. 1952; 117: 500-544Crossref PubMed Scopus (12310) Google Scholar, 2Armstrong C.M Sodium channels and gating currents.Physiol. Rev. 1981; 61: 644-682Crossref PubMed Scopus (343) Google Scholar). Recent studies indicate that ∼12 electronic charges in the sodium channel protein move across the membrane electric field during activation (43Hirschberg B Rovner A Lieberman M Patlak J Transfer of twelve charges is needed to open skeletal muscle Na+ channels.J. Gen. Physiol. 1995; 106: 1053-1068Crossref PubMed Scopus (117) Google Scholar). The novel features of the primary structure of the sodium channel α subunit led directly to hypotheses for the molecular basis of voltage-dependent gating (13Catterall W.A Voltage-dependent gating of sodium channels correlating structure and function.Trends Neurosci. 1986; 9: 7-10Abstract Full Text PDF Scopus (116) Google Scholar, 35Guy H.R Seetharamulu P Molecular model of the action potential sodium channel.Proc. Natl. Acad. Sci. USA. 1986; 508: 508-512Crossref Scopus (403) Google Scholar). The S4 transmembrane segments contain repeated motifs of a positively charged amino acid residue followed by two hydrophobic residues, potentially creating a cylindrical α helix with a spiral ribbon of positive charge around it (Figure 3, left). The negative internal transmembrane electrical field would exert a strong force on these positive charges arrayed across the plasma membrane, pulling them into the cell in a cocked position. The sliding helix (13Catterall W.A Voltage-dependent gating of sodium channels correlating structure and function.Trends Neurosci. 1986; 9: 7-10Abstract Full Text PDF Scopus (116) Google Scholar) or helical screw (35Guy H.R Seetharamulu P Molecular model of the action potential sodium channel.Proc. Natl. Acad. Sci. USA. 1986; 508: 508-512Crossref Scopus (403) Google Scholar) models of gating propose that these positively charged amino acid residues are stabilized in the transmembrane environment by forming ion pairs with negatively charged residues in adjacent transmembrane segments (Figure 3, right). Depolarization of the membrane was proposed to release the S4 segments to move outward along a spiral path, initiating a conformational change that opens the pore. Although this model was quite speculative when it was proposed, its major features have now received direct experimental support from work on both sodium and potassium channels. The first evidence in favor of the S4 segments as voltage sensors came from mutagenesis studies of sodium channels (97Stuhmer W Conti F Suzuki H Wang X Noda M Yahadi N Kubo H Numa S Structural parts involved in activation and inactivation of the sodium channel.Nature. 1989; 339: 597-603Crossref PubMed Scopus (912) Google Scholar). Neutralization of the positively charged residues in the S4 segment of domain I was found to reduce the steepness of voltage-dependent gating, as expected for a reduction in gating charge, and to shift the voltage dependence along the voltage axis (97Stuhmer W Conti F Suzuki H Wang X Noda M Yahadi N Kubo H Numa S Structural parts involved in activation and inactivation of the sodium channel.Nature. 1989; 339: 597-603Crossref PubMed Scopus (912) Google Scholar). Mutations in the positively charged residues in other S4 segments also have strong effects, especially at the fourth position (51Kontis K.J Rounaghi A Goldin A.L Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains.J. Gen. Physiol. 1997; 110: 391-401Crossref PubMed Scopus (119) Google Scholar). Thus, this functional test supports the idea that the positive charges in S4 segments are the gating charges. The proposed outward movement of the S4 segments of sodium channels has been detected directly in cleverly designed mutagenesis and covalent modification experiments. Yang, George, and Horn (112Yang N.B Horn R Evidence for voltage-dependent S4 movement in sodium channel.Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 113Yang N.B George Jr., A.L Horn R Molecular basis of charge movement in voltage-gated sodium channels.Neuron. 1996; 16: 113-122Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar) used sodiu